Identification of an Insulin-Like Receptor Linked to Testis Development in Mud Crabs

The full-length cDNA sequence of Sp-IR was successfully obtained through a combination of 3′ and 5′ RACE techniques and touchdown PCR. The mRNA sequence of Sp-IR in the testis of S. paramamosain was found to be 6538 bp in length (GenBank OQ361826↗). It consisted of a 306 bp 5′ untranslated region (UTR), a 559 bp 3′ UTR, and a substantial open reading frame (ORF) spanning 5673 bp. The ORF encoded a protein consisting of 1890 amino acids (Supplemental S1).

Bioinformatics analysis revealed that Sp-IR belonged to the IR family. The protein could be divided into three distinct regions. The extracellular region comprised a CR domain, two L domains, and three FNIII domains. The transmembrane region was hydrophobic, facilitating its integration into the cell membrane. The intracellular region contained a tyrosine kinase domain, which is crucial for signal transduction and downstream signaling events (Figure 1).

Figure 2 displays the results of multiple sequence alignment of the tyrosine kinase domain. Among the species compared, Sp-IR exhibits the highest similarity with the conserved tyrosine kinase domain of F. chinensis, with a similarity of 81.10%. It also shows a relatively high similarity of 74.32% with the tyrosine kinase domain of S. verreauxi. Comparing Sp-IR with other species, including M. rosenbergii, Bombyx mori, Drosophila melanogaster, Aedes aegypti, and other arthropods, the amino acid sequence similarity exceeds 40% in all cases.

The phylogenetic tree analysis results are presented in Figure 3. Initially, Sp-IR clustered with Fc-IAGR in F. chinensis, Pv-IR in Penaeus vannamei, and Sv-IR in S. verreauxi. Subsequently, it formed a cluster with Mr-IR in M. rosenbergii, indicating a high degree of similarity between these IRs.

The expression pattern of Sp-IR in various tissues of male S. paramamosain at stage II was assessed using RT-PCR. The results revealed that Sp-IR exhibited high expression levels in the testis and AG (Figure 4). However, no transcript of Sp-IR was detected in the other tissues examined.

To investigate the potential involvement of Sp-IR in the reproductive process of male S. paramamosain, the expression profile of Sp-IR was analyzed across three testis’ developmental stages using qRT-PCR. The results demonstrated that Sp-IR exhibited the highest expression level at stage I (Figure 5). However, a significant decrease in Sp-IR expression was observed in both stage II and stage III (p < 0.05).

In studying sex determination and differentiation mechanisms in crustaceans, researchers often rely on homologous marker genes identified in model organisms, such as vasa, Dsx, Sxl, Tra, Dmrt, and Sox-5 genes [23]. Vasa is a well-known marker gene for germ cells due to its specific expression and essential functions in gametogenesis [24]. It has been confirmed in many organisms that vasa play crucial roles in spermatogenesis [25,26,27]. In male S. paramamosain, Sp-vasa was specifically expressed in the testis, and its mRNA was detected exclusively in germ cells [28]. The expression of Sp-vasa gradually increased from spermatogonia to spermatocytes but was absent in spermatozoa, indicating its involvement in spermatogenesis before the formation of spermatozoa. Similarly, the expression pattern of the Dsx gene in the testis of F. chinensis is comparable to that of Sp-vasa [29]. Silencing of Fc-Dsx was shown to inhibit Fc-IAG gene expression, suggesting the involvement of Dsx in IAG-regulated spermatogenesis [29]. Therefore, in this study, we utilized Sp-vasa and Sp-Dsx as indicators of spermatogenesis.

To assess the effectiveness of Sp-IR knockdown in the testis, male crabs at stage I were subjected to RNAi by injecting Sp-IR dsRNA. The results demonstrated a significant reduction in the expression level of Sp-IR after Sp-IR dsRNA injection (p < 0.01). The knockdown efficiency reached up to 85.97% (Figure 6A). Furthermore, the silencing of Sp-IR resulted in a substantial decrease in the expression levels of Sp-vasa (Figure 6B) and Sp-Dsx (Figure 6C) genes in the testis (p < 0.01). In contrast, the injection of GFP dsRNA had no significant effect on the expression levels of Sp-IR, Sp-vasa or Sp-Dsx.

To further investigate the role of Sp-IR in spermatogenesis and testicular development in S. paramamosain, a 22-day RNAi experiment was conducted on male individuals at stage I. At the end of the experiment, the interference efficiency and the expression levels of testis development-related genes were assessed. The results demonstrated a significant down-regulation of Sp-IR expression after 22 days of Sp-IR dsRNA injection compared to the crab saline control group (p < 0.001). The gene knockdown efficiency was calculated to be 75.83% (Figure 7A). Similarly, the expression levels of Sp-vasa (Figure 7B) and Sp-Dsx (Figure 7C) transcripts were significantly reduced following the injection of Sp-IR dsRNA (p < 0.01). In contrast, the injection of GFP dsRNA had no notable effect on the expression levels of Sp-IR, Sp-vasa, or Sp-Dsx transcripts in the testis.

Moreover, the histological analysis revealed that the knockdown of Sp-IR had a detrimental effect on spermatogenesis and testis development in S. paramamosain. In the pre-injection control group, the lumen of the seminiferous tubules contained a substantial number of primary spermatocytes and spermatids, indicating active spermatogenesis (Figure 8A). The crab saline control group and the GFP dsRNA group showed similar proportions of the four types of cells compared to the pre-injection control group, with only a few spermatids transitioning into spermatozoa (Figure 8B,C). In contrast, the injection of Sp-IR dsRNA resulted in irregularly shaped seminiferous tubules and a significant reduction in their volume. Large vacuoles were also observed in the testis. Furthermore, most of the seminiferous tubules were occupied by spermatogonia and primary spermatocytes, with only a small number of secondary spermatocytes present. No spermatids or mature sperm were observed (Figure 8D).

Mud crabs (Scylla paramamosain) were purchased from a local fisheries wholesale market in the Si-Ming District of Xiamen (Fujian, China). The male individuals selected for the experiments exhibited healthy appendages and good vitality and were at the intermolt stage. Prior to the start of the experiments, the crabs were acclimated in tanks containing filtered seawater at a temperature of 27 ± 2 °C and salinity of 27 ± 0.5 ppt for 5 days and fed with fresh Manila clams (Ruditapes philippinarum) daily. Any uneaten bait was promptly removed, and the tanks were replenished with fresh seawater containing sufficient oxygen the following morning.

The development of S. paramamosain testis can be divided into five stages, which closely align with the staging system of the Chinese mitten crab Eriocheir sinensis [38,39]. In stage I, the testis is transparent, and the seminiferous tubules are relatively small, measuring approximately 105 μm in diameter. This stage is characterized by a high proportion of spermatogonia and primary spermatocytes, with occasional presence of secondary spermatocytes and spermatids. In stage II, the testis appears semitransparent with white coloration. The seminiferous tubules are larger than in stage I, with a diameter of around 210 μm. Most of the germ cells at this stage are spermatocytes. In stage III, the testis takes on a white or milk-white coloration, and the seminiferous tubules further increase in size, measuring approximately 4–6 mm in diameter. Spermatids become the prominent germ cells at this stage. In stage IV, mature sperms become predominant in the testis, and the lumen of the seminiferous tubules continues to expand. In stage V, the testis exhibits a faint yellow color, the seminiferous tubules are devoid of sperm, and the germinal zone undergoes degradation, shrinkage, or even disappear.

Since the differentiation of spermatogonia and spermatocytes occurs before stage IV, the crabs in stages I to III were selected to analyze Sp-IR expression. The crabs used for the analysis had the following characteristics: stage I (body weight: 35.67 ± 4.82 g, carapace width: 4.20 ± 0.51 cm), stage II (body weight: 108.24 ± 15.68 g, carapace width: 6.50 ± 0.58 cm), and stage III (body weight: 274.52 ± 18.73 g, carapace width: 8.70 ± 0.45 cm). The crabs were anesthetized by placing them on ice, and testis samples were collected from males at each stage (n = 4–5) for Sp-IR expression profile analysis using real-time quantitative PCR (qRT-PCR). The samples were then either immediately used for RNA extraction or stored in liquid nitrogen.

Total RNA was extracted from the samples using TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The concentration and quality of the extracted RNA were determined using a Q6000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The integrity of the RNA was evaluated by performing 1.5% (w/v) agarose gel electrophoresis. First-strand cDNA synthesis was conducted for subsequent experiments using RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific).

A partial cDNA sequence encoding an insulin-like receptor was initially identified from the transcriptome database of S. paramamosain. Subsequently, the full-length sequence was amplified using specific primers IR-F and IR-R. To obtain the complete sequence of Sp-IR, 3′-RACE and 5′-RACE experiments were performed using the SMARTer™ RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) following the manufacturer’s protocol. The PCR products obtained were separated by 1.5% (w/v) agarose gels, purified using an agarose gel purification and extraction kit (Aidlab, Beijing, China), and ligated into a pMD19-T vector (Takara, Ohtsu, Japan) for further sequencing. Then, the full-length cDNA sequence of Sp-IR was assembled from the obtained sequencing results. The primers utilized in this study are listed in Table S1.

The open reading frame (ORF) and amino acid sequence of Sp-IR were predicted using the ORF finder tool. The conserved domains of Sp-IR were predicted using the SMART website (http://smart.embl-heidelberg.de/↗; accessed on 7 November 2022), while the transmembrane domain of Sp-IR was predicted using an online website (http://www.cbs.dtu.dk/services/TMHMM/↗, accessed on 7 November 2022). To determine the presence of a protein signal peptide, the amino acid sequence was submitted to the SignalP 5.0 Server (http://www.cbs.dtu.dk/services/SignalP/↗; accessed on 7 November 2022). To assess the homology of the amino acid sequence of Sp-IR with those of other species, BlastX homology searches were performed using the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi↗; accessed on 8 November 2022). Multiple sequence alignment of the tyrosine kinase domain was conducted using Cluster X2.0 and GeneDoc2.7.000 software. Phylogenetic trees were constructed using the neighbor-joining (NJ) method implemented in MEGA 7.0.26 software, with bootstrap sampling repeated 1000 times to assess the reliability of the tree.

To determine the tissue distribution of Sp-IR in male mud crabs at stage II, reverse transcription PCR (RT-PCR) was performed. The cDNA templates were generated from 11 different tissues, including the eyestalk ganglion, cerebral ganglion, thoracic ganglion, heart, muscles, hepatopancreas, stomach, epidermis, testis, androgenic gland, and Y-organ. PCR was performed using the following conditions: an initial denaturation step at 95 °C for 3 min, followed by 36 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. The final extension step was performed at 72 °C for 10 min. Negative controls, using water as a template, were included to ensure the absence of contamination. The amplification of β-actin was used as the internal control for normalization purposes. After amplification, the PCR products were visualized by electrophoresis on a 1.5% agarose gel and imaged using a UV gel imager (Thermo Scientific, USA). The experiment was repeated thrice.

To investigate the impact of Sp-IR on testis differentiation and development in the mud crab S. paramamosain, a long-term RNAi experiment was conducted on male crabs at stage I. The crabs used in the experiment had a body mass of 37.56 ± 3.68 g and were temporarily housed in the laboratory for 5 days. Before the injection, six individuals were randomly selected as the pre-injection control group (n = 6). The remaining crabs were divided into three groups (n = 30). The control group received an injection of 100 μL of crab saline. The other two groups were injected with Sp-IR dsRNA and GFP dsRNA (1 μg/g body mass) prepared in 100 μL of crab saline, respectively. The injections were performed once every five days, and on day 22 of the experiment, approximately 48 h after the fourth injection, the crabs were anesthetized by placing them on ice and then euthanized. Testis samples were dissected from the crabs for gene expression analysis of Sp-IR, Sp-vasa, and Sp-Dsx, using techniques such as qRT-PCR. Additionally, histological analysis of the testis was conducted through hematoxylin and eosin staining.

The relative expression levels of the target genes were determined using qRT-PCR. The reactions were carried out on a 7500 Fast Real-Time PCR Detection System (Applied Biosystems, Carlsbad, CA, USA) with a total reaction volume of 20 μL. Each reaction included 2 μL of diluted cDNA, 10 μL of 2× PCR Master Mix with SYBR GREEN (Thermo Scientific, Vilnius, Lithuania), 0.5 μL each of forward and reverse primers (1 mM), and 7 μL of water. The thermal profile for the qRT-PCR reaction consisted of an initial incubation at 95 °C for 3 min, followed by 40 cycles of amplification with the following conditions: denaturation at 95 °C for 10 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. Each sample was analyzed in triplicate, and the β-actin gene was simultaneously amplified as an internal control to normalize the data. The primers used for the qRT-PCR are listed in Table S1. The relative transcript abundance of the target genes was calculated using the 2^−ΔΔCt method.

The data are presented as mean ± SEM (standard error of the mean). Statistical analysis was performed using the SPSS v20.0 software. Levene’s test was performed to examine the homogeneity of data variance. If the variance was found to be homogeneous, significance analysis was performed using one-way analysis of variance (ANOVA), followed by Duncan’s test or Dunnett multiple comparison test. Statistical significance was set at p < 0.05.

The data presented in this study are openly available in GenBank [OQ361826↗].